Method for the enzymatic production of a curing agent and its fluid state

ABSTRACT

The invention relates to a method for the enzymatic production of a curing agent in its fluid state, e.g. liquid, comprising, in free phase, at least one oxygenated chemical species. Said method consists in bringing into contact at least one enzymatic catalysts agent, comprising at lease one peroxidase-type enzyme; an oxidizable substrate in aqueous phase that can be oxidized by the action of an oxygen donor, by catalysis by said enzymatic catalysis agent, generating said oxygenated chemical species in free phase; and said oxygen donor. The inventive method is characterized in that: e) an aqueous reaction bath is formed comprising, in addition to the oxidizable substrate and the oxygen donor, said enzymatic catalysis agent in divided solid phase, but in free phase, distributed is said bath, which may be set in motion; f) the aqueous reaction bath is separated into a fraction enriched with the enzymatic catalysis agent in divided solid phase and a fraction free from said catalysis agent, from which the curing agent is obtained.

The present invention relates to generating a concentrated andcontinuous flow, in liquid phase, of oxygenated chemical species andoxidized substrates, thereby making it possible to obtain solutionswhich can be used, for example, for washing, decontaminating andsterilizing different alimentary products, including water, and alsoindustrial materials, as well as for depolluting and purifying fluidsand for preparing alimentary, pharmaceutical and cosmetic products.

The use of natural antimicrobial enzyme systems, such as theoxidoreductases, for example the lactoperoxidase system, for thispurpose is known and a large number of applications have been described.

The properties of this enzyme system have been studied, in particular,in “The lactoperoxidase system chemistry and biological significance”(1985) Marcel Dekker, Inc, New York, Chap. 8 pp 143-178.

In outline, this lactoperoxidase/thiocyanate/hydrogen peroxideantimicrobial system comprises three components:

-   -   an enzyme: lactoperoxidase,    -   an oxidizable substrate: the thiocyanate ion (SCN⁻)    -   an oxygen donor: hydrogen peroxide.

In this system, and in liquid medium, the lactoperoxidase catalyses thethiocyanate oxidation reaction.

In the presence of a sufficient quantity of hydrogen peroxide and underthe correct pH conditions, the oxidation reaction continues towardsoxyacid derivatives which are even more oxidized.

In a non-limiting manner, the oxygenated chemical species which areobtained, either on their own or in a mixture, are the hypothiocyaniteion OSCN⁻, the O₂SCN⁻ and O₃SCN⁻ ions, the superoxide O₂ ⁻ and trioxideO₃ ⁻ an ions, the hydroxyl ion OH⁻, nitric oxide NO, dinitrogen trioxideN₂O₃, nitrogen dioxide NO₂, peroxynitrite ONO₂, hydroperoxynitrileONHO₂, sulfur dioxide SO₂, sulfur trioxide SO₃, sulfurous acid HSO₃ andhypochlorous acid HOCl.

The abovementioned oxygenated chemical species are known to havebacteriostatic and bactericidal effects, in particular in regard to alarge number of microorganisms such as bacteria, for example thePseudomonae, the Enterobacteriaceae, such as E. coli, Salmonella, theListeria or Campylobacter, sporulated forms and protozoa, viruses,yeasts or fungi.

As a result of the action of the oxygenated chemical species obtained,in particular the oxidized thiocyanate ions such as OSCN⁻, O₂SCN⁻ andO₃SCN⁻, which are able to interact with the components of cell membranesor to oxidize chemical pollutants, this antimicrobial system can also beused for decontamination.

In the presence of a hydrogen peroxide-supplying substrate, which can behydrogen peroxide itself or, for example, a metal peroxide or asupplementary enzyme system which produces hydrogen peroxide, with thissupplementary enzyme system being, for example, an oxidoreductasetogether with an oxidizable substrate and oxygen, such as theglucose/glucose oxidase system in aqueous medium, it is possible to usethe complete system and, in outline, the reaction properties of such anantimicrobial system comprise three steps:

-   -   the production of hydrogen peroxide by the hydrogen peroxide        supplier,    -   the thiocyanate oxidation reaction,    -   decontamination by the action of the oxygenated chemical species        obtained.

A large number of applications are described.

For example, WO-A-8707838 describes a process for packaging, in dryform, an antibacterial composition containing lactoperoxidase,thiocyanate and a native oxygen donor with a view to its subsequent use.

U.S. Pat. No. 5,403,450 discloses the use of units in whichoxidoreductases are immobilized for the purpose of converting substanceswhich are capable of being oxidized and which are present as pollutantsin water.

JP58152486 teaches, for example, the immobilization of enzymes onpolymer particles for repeated use.

Systems for immobilizing enzymes or enzyme systems on reactor walls oron films, beads and other supports comprising a substantial specificarea are also known.

However, none of these uses is satisfactory due to the excessiveconsumption of enzymes which they entail:

either due to their packaging, for example in dry form, which involves ause by powdering the products to be treated, and therefore quantities ofenzyme which are proportional to the areas and to the volumes ofproducts to be treated,

or due to the low reaction yields of the immobilized enzymes,

or due to the rapid breakdown of the oxygenated chemical species whichare generated.

The process according to the invention makes it possible to solve allthe abovementioned drawbacks by making it possible to produce atreatment agent in the fluid, for example liquid, state, which agentcomprises, in the free state, at least one stable oxygenated chemicalspecies with high yield and exhibiting substantial endurance.

The invention relates to:

a process for enzymically producing a treatment agent in the fluid, forexample liquid, state, comprising, in the free state, at least oneoxygenated chemical species, by bringing into contact at least:

-   -   one agent of enzymic catalysis, comprising at least one enzyme        of the peroxidase type,    -   and one substrate which can be oxidized in aqueous phase and        which is capable of being oxidized by the action of an oxygen        donor, by means of catalysis by said agent of enzymic catalysis,        thereby generating said oxygenated chemical species in the free        state,        -   said oxygen donor,            according to which:

-   a) an aqueous reaction bath is formed, which bath comprises, in    addition to the oxidizable substrate and the oxygen donor, said    agent of enzymic catalysis in the solid and divided phase, but in    the free state, which agent is distributed in said bath, which    latter may possibly be set in motion,

-   b) the aqueous reaction bath is separated into a fraction which is    enriched in agent of enzymic catalysis in solid and divided phase    and into a fraction which lacks said agent of catalysis and from    which the treatment agent is obtained.

The oxygen donor is preferably a peroxide such as hydrogen peroxide.

An oxygenated chemical species in the free state is understood as beinga chemical species in the ionic state whose dissociation constant, atthe pH of the resulting solution, displaces the equilibrium of thedissociation reaction towards the existence, in the free state, of saidoxygenated chemical species.

The invention affords the following variants:

the oxidizable substrate, possibly in aqueous phase, is introduced intothe aqueous reaction bath.

the agent of enzymic catalysis, in the state of a solid and dividedphase or in liquid phase, is introduced into the reaction bath.

the agent of enzymic catalysis is discharged from the reaction bath.

the process is performed continuously or discontinuously.

aggregates of solid particles, which are inert vis-á-vis the agent ofenzymic catalysis, are formed in the aqueous reaction bath, with saidaggregates comprising or incorporating, in the free state, said agent ofcatalysis, and said aggregates being distributed in the aqueous reactionbath and by means of which the said bath is separated, during step (b),into a fraction which is enriched in aggregate and a fraction which isdevoid of aggregate and from which the treatment agent is obtained.

the aggregates are floccules and a flocculating agent, for example ananionic or cationic flocculating agent, is introduced into the reactionbath.

the aggregates are coagulates and a coagulating agent is introduced intothe reaction bath.

a thickening agent is introduced into the bath.

the agent of enzymic catalysis in solid phase is suspended in thereaction bath, in emulsion form, and, where appropriate, an emulsifyingagent is introduced into said bath.

the agent of catalysis comprises a microorganism which is expressing atleast one enzyme of the peroxidase type.

a pH-correcting agent is introduced into the bath.

an oxygen donor in the form of a supplementary enzyme system whichproduces hydrogen peroxide is introduced into the bath and the agent ofenzymic catalysis comprises, in addition to the enzyme of the peroxidasetype, an enzyme of the oxidoreductase type.

An aggregate is understood as being any formulation which makes itpossible to maintain the agent of enzymic catalysis in solid and dividedphase, but in the free state, in the reaction medium by means of addingcoagulant, flocculant or emulsifier, in the presence or absence of athickener, but which makes it possible to isolate them from saidreaction medium at the end or the reaction and to recycle them.

The free state is understood as meaning a state of suspension in theaggregates without the formation of an ionic or covalent bond betweenthe agent of catalysis and said aggregating agents.

In one implementation variant, the aggregates are floccules.

Floccules are understood as meaning any aggregate of particles which arechemically inert vis-á-vis the enzyme or the enzymic system and whichcontain said enzyme without immobilizing it, which aggregate can beobtained by adding a cationic or anionic flocculant, after coagulationor without preliminary coagulation, and which is incorporated with orwithout a pH-correcting agent.

The flocculants which are used are selected from polymeric an ionic orcationic flocculants, such as polysaccharides, anionicheteropolysaccharides or polyacrylamines.

Advantageously, the flocculant's are added to the reaction medium inproportions varying from 0.1 10⁻⁶ to 10 g/l of reaction medium.

In one implementation variant, the aggregates are coagulates.

A coagulate is understood as meaning any aggregate of particles whichare inert vis-á-vis the enzyme or the enzyme system and which containsaid enzyme without immobilizing it, which aggregate can be obtained byadding a coagulating agent as previously defined.

Advantageously, the coagulants are added to the reaction medium inproportions varying from 0.1 10⁻⁶ to 10 g/l of reaction medium.

The aggregates can be formed by consecutively adding a coagulant and aflocculant; in this way, a coagulation step precedes the flocculationstep.

When the flocculation step is preceded by a coagulation step, the latteris effected by introducing a coagulating agent which is selected, forexample, from the salts of aluminum or iron, such as: aluminum sulfate,aluminum chloride, sodium aluminate, aluminum polyhydroxychloride,aluminum polyhydroxysulfate, aluminum polyhydroxychlorosulfate, basicaluminum poly-chlorosulfate, aluminum polyhydroxychlorosilicate,aluminum fluorosulfate, ferrous sulfate, ferric sulfate, ferricchloride, ferric chlorosulfate, soda or homopolymers ofdimethyldiallylammonium chloride.

The invention also relates to a group of reagents for enzymicallyproducing a treatment agent in the fluid state which comprises, in thefree state, at least one oxygenated chemical species, which furthermorecomprises an agent of enzymic catalysis and an aggregating agent inaqueous phase and/or a flocculating agent in aqueous phase and/or acoagulating agent in aqueous phase.

In one embodiment, an organic or inorganic thickening agent, which ischemically compatible with the aggregate type which it is desired touse, is added to the mixture.

According to the implementation variants, this thickening agent isintroduced into the bath at the same time as introducing the agent ofenzymic catalysis or after the aqueous reaction bath has been formed.

This thickening agent is selected from clays, kaolin, silica orsilicates or any other compatible inorganic agent which promotesentrainment of the enzyme.

Advantageously, the thickening agent is added to the reaction medium inproportions varying from 0.1 to 100 g/l of reaction medium.

The invention also relates to a group of reagents in unhydrated form forenzymically producing a treatment agent in the fluid state whichcomprises, in the free state, at least one oxygenated chemical species,which comprises at least one agent of enzymic catalysis and a thickeningagent.

Advantageously, the group of reagents in unhydrated form comprises from0.005 to 10% of agent of enzymic catalysis and from 90 to 99.995% ofthickening agent, for example from 5 g to 400 g of lactoperoxidase indry form and from 1000 g to 5000 g of clay for a reactor having aserviceable volume of 2 m³.

The group of reagents is employed by introducing it into the reactor atthe rate of from 0.2 to 10 g/l of serviceable volume.

In another embodiment, an emulsifying agent is added to the mixture.

Emulsion is understood as meaning any suspension which contains anenzyme without immobilizing it and which can be obtained by addingemulsifiers such as fats or soybean lecithin or hydrocarbons.

In particular embodiments, the pH of the aggregate dispersion medium canbe stabilized or corrected by adding a pH-correcting agent which will beselected from inorganic or organic acids or bases.

The agents of enzymic catalysis are enzymes which are free, chemicallyisolated or associated among themselves and which are obtained fromisolated microorganisms, cells of vegetable or animal origins orpolymicrobial flora. They are, in particular, peroxidases and anynatural, selected or genetically modified microorganism which producesenzymes or generates enzymic activity.

The peroxidases which can be used in the present invention compriseperoxidases of vegetable origin, such as horseradish peroxidase orsoybean peroxidase, or cereal NADPH nitrate oxidoreductase, or of animalor human origin, such as saliva peroxidase, lactoperoxidase,myeloperoxidase and eosinophil peroxidase.

These peroxidases can be extracted and/or isolated from naturalsubstances such as milk or saliva or be produced using natural orchemical processes which are well known to the skilled person. Theseperoxidases can also be produced by means of recombinant techniques,with these latter also being well known to the skilled person.

The agents of enzymic catalysis are preferably selected fromperoxidases.

In a preferred embodiment, the peroxidase is a lactoperoxidase.

Advantageously, the enzymes are added to the reaction medium inproportions varying from 0.02 to 10 g/l of reaction medium.

The oxidizable substrates are selected from the group consisting ofnegatively charged halogens and their derivatives and negatively chargedpseudohalogens and their derivatives. In this present case, the termnegatively charged halogen refers to certain chemical elements in theform of anions which belong to Group VII of the Periodic Table of theElements and which are, for example, bromides, Br⁻, chlorides, Cl⁻ oriodides, I⁻.

The pseudohalogens are, for example, selected from the group consistingof thiocyanate ions, bisulfite ions, hydrosulfite ions, metabisulfiteions and nitrite and/or hypochlorite ions.

The oxidizable substrates will be selected, by preference and inaccordance with the peroxidase employed, from sodium thiocyanate (NASCN)or potassium thiocyanate (KSCN), sodium bisulfite (NaHSO₃), sodiumhydrosulfite (Na₂S₂O₄), sodium metabisulfite (Na₂S₂O₅), sodium nitrite(NaNO₂) or potassium nitrite (KNO₂), sodium hypochlorite (NaOCl) andpotassium iodide (KI).

For example, if the peroxidase is:

a saliva peroxidase, either thiocyanate ions or iodide ions and/or theirmixtures will be used as the oxidizable substrate,

a lactoperoxidase, either thiocyanate ions or iodide ions and/or theirmixtures will be used as the oxidizable substrate,

a myeloperoxidase, either thiocyanate ions or iodide ions or chlorideions and/or their mixtures will be used as the oxidizable substrate,

a horseradish peroxidase, either chloride ions or iodide ions and/ortheir mixtures will be used as the oxidizable substrate,

a peroxidase of vegetable origin, either thiocyanate ions or bromideions or chloride ions and/or their mixtures will be used as theoxidizable substrate.

In the presence of peroxide, for example oxygen peroxide, the action ofthese enzyme systems results in the production, in particular, ofhypothiocyanite ions or hypohalide ions.

Advantageously, the combined enzyme substrates are added to the reactionmedium in proportions varying from 0.05 mM to 15 mM per liter ofreaction medium.

The oxygen donor according to the present invention can be hydrogenperoxide or any inorganic peroxide such as the metal peroxides, forexample magnesium peroxide or sodium peroxide, or organic peroxides,such as benzyl peroxide, or urea peroxide, but also peracetic acid,potassium permanganate and the percarbonates. In a general manner, anychemical compound which is capable of producing hydrogen peroxide can beused.

Advantageously, the oxygen donors are added to the reaction medium inproportions varying from 0.05 mM to 15 mM per liter of reaction medium.

When the oxygen donor is in the form of a supplementary enzyme systemwhich produces oxygen peroxide, it comprises an oxidizable substrate andan enzyme, for example of the oxidoreductase type, which is specific forthis substrate. Thus, when the enzyme systems employed areoxidoreductases, an oxidizable substrate, alone or in combination, withthis substrate being selected from substances such as glucose, lactoseor xanthine, is added to the medium in order to effect the step ofproducing the hydrogen peroxide.

The following enzyme systems will be mentioned by way of example:glucose oxidase/glucose, galactose oxidase/galactose, urateoxidase/urate, choline oxidase/choline, glycine oxidase/glycine,glutamate oxidase/glutamate and alcohol oxidase/alcohol.

In the presence of oxygen and water, these enzyme systems are able toproduce hydrogen peroxide, which will then be used as oxygen donor inthe enzyme system of the process according to the invention.

Alternatively, this oxygen donor can be selected from microorganisms,such as Streptococcus and/or Lactobacillus, which are able to producehydrogen peroxide.

The peroxidases and/or oxidoreductases which are employed and which maybe mentioned by way of example are:

enzymes of vegetable origin such as horseradish peroxidase (E.C. No.1.11.1.7) or soybean peroxidase, or cereal NADPH nitrate oxidoreductase(E.C. No. 1.6.6.1),

enzymes of fungal origin, such as glucose oxidase (E.C. No. 1.1.3.4),catalase (E.C. No. 1.11.1.6), betagalactosidase (E.C. No. 3.2.1.23) andAspergillus NADPH nitrate oxidoreductase (E.C. No. 1.6.6.2),

enzymes of bacterial origin, such as Enterococcus NADH peroxidase (E.C.No. 11.1.1), Vibrio NADPH oxidoreductase (E.C. No. 1.6.99.3),Colibacillus nitrate reductase (E.C. No. 1.9.6.1), Pediococcus lacticdismutase oxidase (E.C. No. 1.1.3.2), Colibacillus superoxide dismutase(E.C. No. 1.15.1.1), Arthromyces peroxidase (E.C. No. 1.11.1.1) andColibacillus betagalactosidase (E.C. No. 3.2.1.23),

enzymes of animal origin, such as milk xanthine oxidase (E.C. No.1.1.3.22), milk lactoperoxidase (E.C. No. 1.11.1.7), leucocytemyeloperoxidase (E.C. No. 1.11.1.7), nervous tissue nitric oxidesynthetase (E.C. No. 1.14.13.39), erythrocyte super-oxide dismutase(E.C. No. 1.15.1.1) and hepatocyte sulfite oxidase (E.C. No. 1.8.3.1).

Implementation of step b) of the process according to the invention,that is to say the step of separating the aqueous reaction bath into afraction which is enriched in agent of enzymic catalysis in solid anddivided phase and a fraction which is devoid of said agent of catalysisand from which said treatment agent is obtained, is effected by means ofseparating and recovering the aggregates or the emulsions. The meanswhich are used and which will be mentioned by way of example are themeans which are classically used such as decantation, flotation andcentrifugation, in the case of emulsions, and sedimentation, frontal ortangential filtration or a centrifugation, in the case of flocculates orcoagulates, and/or cyclonic separation.

The process according to the invention makes it possible to obtain atreatment agent in the fluid, for example liquid, state, which agentcomprises, in the free state, at least one stable oxygenated chemicalspecies.

More specifically, the stable oxygenated chemical species is thehypothiocyanite ion (OSCN⁻).

The invention also relates to a treatment agent in the fluid, forexample liquid, state, which agent comprises, in the free state, atleast one oxygenated chemical species which is stable for more than 10hours.

It relates, more specifically, to a treatment agent in the fluid, forexample, liquid state which comprises, in the free state, at least thehypothiocyanite ion, which is stable for more than 10 hours.

The process according to the invention makes it possible to producelarge quantities of treatment agent solution according to the invention,which solution possesses biocidal properties which can be used forcleaning, washing and disinfecting materials, machines, implements,textiles, containers and pipework and agroindustrial premises and plantsor hospitals and care centers. The process also makes it possible toprepare solutions which are intended for formulating cosmetic andpharmaceutical products which are intended for human and/or animalhealth and alimentary products.

The process also makes it possible to produce washing solutions fordecontaminating the surfaces of alimentary products such as fruit,vegetables and leaves, and also animal products.

These biocidal solutions will be able to be used by means of bathing,spraying, injection or nebulization.

It will also be possible to use these solutions as constituents of aproduct mixture, for example water for reconstituting fruit juicefollowing dehydration or concentration.

The process also makes it possible to sterilize and purify water whichis intended for producing water for beverages for human or animalconsumption, or thermal spring water, swimming pool water or bath water.

The process according to the invention also makes it possible to treatpolluted water, wastewater or industrial effluents and, indeed, gaseouseffluents, by circulating them in the liquid.

The chemical contaminants, for example nitrates or phosphates, are thusoxidized, as are the organic pollutants; it will be possible for thebreakdown to be total or partial, depending on the flowrate and theconcentrations of oxidoreductase.

The implementation of the process according to the invention, that is tosay a process for enzymatically producing a treatment agent in thefluid, for example liquid, state, which agent comprises, in the freestate, at least one oxygenated chemical species, by bringing intocontact at least one agent of enzymic catalysis, comprising at least oneenzyme of the peroxidase type, an oxygen donor and a substrate which iscapable of being oxidized by said agent of enzymic catalysis, therebygenerating said oxygenated chemical species in the free state, iseffected in a reactor (FIG. 1) which consists of a compartmentalizedtank, which can be closed partially or completely, which is made ofmetal or a synthetic material and which is provided with a loadingaperture (1) and overflows and/or partitions in the form of siphons,permitting passage from one compartment to the next (2).

The reactor according to the invention is made up of three or fourcompartments, two or three of which are stirred continuously:

the first (3) is intended to receive the agent of enzymic catalysis andoptionally the coagulant and the thickening agent; it is stirred at highspeed,

the second (4) optionally receives the flocculant and optionallyreceives the pH-correcting agent; it is stirred at low speed,

the third, which is supplied with oxidizable substrate as well as oxygendonor, is the site of the desired enzyme reaction and is also stirredslowly,

in the fourth compartment, the aqueous reaction bath is separated, intoa fraction which is enriched with agent of enzymic catalysis in solidand divided phase and a fraction which is devoid of said agent ofcatalysis and from which said treatment agent is obtained, on a lamellarsedimentor which comprises a low supply point (6), an overflow (7) whichis connected to the outlet, which is located just below the water stream(8), and a low point for dynamically extracting the sedimented solidmatter (9) with a view to discharging it (10) or recovering it with aview to recycling (11).

In an implementation variant, the process according to the invention,that is to say a process for enzymically producing a treatment agent inthe fluid, for example liquid, state, which agent comprises, in the freestate, at least one oxygenated chemical species, is implemented, bybringing into contact at least one agent of enzymic catalysis,comprising at least one enzyme of the peroxidase type, an oxygen donorand an oxidizable substrate, which is capable of being oxidized by saidagent of enzymic catalysis, thereby generating said oxygenated chemicalspecies in the free state, in a reactor (FIG. 2) which consists of acompartmentalized tank, which can be partially or completely closed,which is made of metal or of a synthetic material, and which is providedwith a loading aperture (1) of overflows permitting passage from onecompartment to another, and which comprises three compartments, thefirst two of which are stirred continuously.

The first, into which the agent of enzymic catalysis and the emulsifyingagent are introduced, is stirred rapidly,

the second, to which the oxidizable substrate is supplied and which isthe site of the desired enzymic reaction, is stirred at low speed;

the third compartment is a recovery vat (5) which enables the emulsion,of the solution comprising at least one oxygenated chemical species inthe free state and residual substrates, to be continuously pumped towarda separation unit, which can be a coalescer, a flotator, a centrifuge(6), a filter or a cyclone.

Applying the process according to the invention to the production of asolution of oxygenated chemical species in solution, by means of usinglactoperoxidase, in flocculate form, in the presence of from 0.2 to 0.5mM H₂O₂ and from 0.4 to 1 mM KSCN, results in an aqueous solution whichcontains between 0.05 and 0.35 mM of oxygenated chemical species.

When used for washing and decontaminating lettuces by means ofconsecutive sprays and baths at 10° C., this solution enables thepopulation of bacterial contaminants, such as Pseudomonas bacteria (10⁵cfu/ml) to be reduced significantly (from 2 to 5 logs on average), andListeria bacteria (10⁵ cfu/ml) to be reduced significantly by from 1 to2 logs, as compared with untreated control lettuces, with a contact timeof the order of 10 minutes.

EXAMPLES

In a reactor such as that described in FIG. 2, 0.25 g/l lactoperoxidaseare introduced at the same time as 10 g/l clay and 0.55 ml of coagulant,while variable quantities of KSCN and H₂O₂, as shown in Table 1 below,are introduced following stirring and passage into the reactioncompartment.

TABLE 1 1 2 3 4 KSCN (mM) 0.5 0.6 0.7 0.8 H₂O₂ (mM) 0.3 0.4 0.5 0.6

Following reaction, samples are removed for measuring enzymic activityand the resulting level of free chemical species.

The hypothiocyanite ions (OSCN⁻) are able to react with the sulfhydrylgroups of a 5,5′-dithiobis (2-nitrobenzoic acid) molecule which haspreviously been reduced in the presence of an excess of sodiumborohydride.

This reduced molecule absorbs light at a wavelength of 412 nm.

When the hypothiocyanite ions (OSCN⁻) oxidize the sulfhydryl groups, theabsorbance at 412 nm decreases in proportion to the quantity of ionswhich are present in the sample, thereby enabling them to be quantified.

The thiocyanate ion (SCN⁻) is able to react with FeCl3 in acid medium inorder to form a colored product the quantity of which is proportional tothat of the thiocyanate present and can be measured by photometry at awavelength of 450 nm.

In order to measure the quantity of thiocyanate which is present in asample, it is necessary to produce a calibration range.

TABLE 2 1 2 3 4 OSCN⁻ ΔOD_(412nm) 0.855^(1/2) 0.999^(1/2) 1.250^(1/2)1.320^(1/2) [OSCN] mM 0.190 0.220 0.275 0.290 Initial OD_(412nm) 0.2420.252 0.279 0.310 SCN⁻ [SCN⁻] mM 0.67 0.70 0.77 0.86 Residual OD_(412nm)0.184 0.186 0.210 0.237 SCN⁻ [SCN⁻] mM 0.51 0.52 0.58 0.66 pH 6.66 6.626.63 6.59 Coagulation quality 3/5 3/5 3/5 2.5/5

The initial enzyme activity in the reactor is checked in assay No. 4.

This is done using 50 μl of coagulate and corresponds to:ΔOD _(405nm)/10 seconds=1.023The enzyme activity I measured by colorimetry at 405 nm using achlorogenic substrate, i.e. 2,2′-azino-bis or ABTS.

Lactoperoxidase catalyses the oxidation of the ABTS in the presence ofhydrogen peroxide. The oxidized ABTS molecule has the property ofabsorbing at 405 nm, thereby making it possible to measure the activityof an enzyme solution by following the quantity of oxidized ABTS whichis produced (proportional to the OD_(405nm) in accordance with theBeer-Lambert law) per unit of time, with the absorbance being measuredcontinuously at 405 nm.

Monitoring the Stability of the Solution.

The methods described above are used to monitor the change in theconcentration of hypothiocyanite ion, i.e. OSCN⁻, in solutions leavingthe reactor, the results of which are obtained are illustrated in FIGS.3 and 4, which depict the curve for the changes in the concentration ofhypothiocyanite ion, OSCN⁻, in the water leaving the reactor over aperiod of 1 day (FIGS. 4) and 4 days (FIG. 3).

The results which are obtained show (see FIG. 4) that the concentrationof OSCN⁻ changes from 600 mM to 500 mM after 10 hours and that itsconcentration has only fallen by 50% after 20 hours.

Over a period of 4 days, a curve is obtained (see FIG. 3) which shows,after 80 hours, a residual concentration of hypothiocyanite ion, OSCN⁻,which is equal to 16% of the initial concentration

1. A process for enzymatically producing a treatment agent in a fluidstate that lacks an enzymatic catalyst, the treatment agent comprisingat least one oxygenated chemical species in a free state, by bringinginto contact in an aqueous reaction bath a mixture comprising: anenzymatic catalyst comprising a peroxidase; a flocculating agentselected from the group consisting of polysaccharides, anionicheteropolysaccharides and polyacrylamines, and/or a coagulating agentselected from the group consisting of salts of aluminum and salts ofiron; an oxygen donor; and a substrate; the process comprising: a)forming aggregates of solid particles that are inert to the enzymaticcatalyst, wherein the aggregates comprise or incorporate the enzymaticcatalyst, and the aggregates are distributed in the aqueous reactionbath, wherein the enzymatic catalyst is in a solid and divided phase,but in a free state, in the aqueous reaction bath; b) generating anoxygenated chemical species in the free state by allowing the enzymaticcatalyst to catalyze oxidation of the substrate by the oxygen donor; andc) separating the aqueous reaction bath into a first fraction enrichedwith the enzymatic catalyst in the solid and divided phase, and a secondfraction that lacks the enzymatic catalyst and contains the treatmentagent, wherein the first fraction is enriched with the aggregates, andthe second fraction is devoid of the aggregates.
 2. The process asclaimed in claim 1, wherein the enzymatic catalyst is introduced intothe reaction bath, in the solid and divided phase.
 3. The process asclaimed in claim 1, wherein the enzymatic catalyst is introduced intothe reaction bath as a solid and divided form entrained in a liquidphase.
 4. The process as claimed in claim 1, wherein the oxidizablesubstrate is introduced into the aqueous reaction bath as an aqueousphase.
 5. The process as claimed in claim 1, wherein the oxygen donor isintroduced into the aqueous reaction bath as an aqueous phase.
 6. Theprocess as claimed in claim 1, wherein, following the separation step(c), the enzymatic catalyst is discharged from the aqueous reactionbath.
 7. The process as claimed in claim 1, wherein said process isperformed continuously or discontinuously.
 8. The process as claimed inclaim 1, wherein the enzymatic catalyst is suspended, in solid phase, inthe aqueous reaction bath in the form of an emulsion and an emulsifyingagent is introduced into said bath.
 9. The process as claimed in claim1, wherein, during step (a), a thickening agent is introduced into theaqueous reaction bath.
 10. The process as claimed in claim 9, whereinthe thickening agent is selected from clays, kaolin, silica orsilicates.
 11. The process as claimed in claim 1, wherein apH-correcting agent is introduced into the bath.
 12. The process asclaimed in claim 1, wherein an oxygen donor in the form of asupplementary enzyme system which produces hydrogen peroxide isintroduced into the bath and the enzymatic catalyst further comprises anoxidoreductase.
 13. The process as claimed in claim 1, wherein theperoxidase is a lactoperoxidase.
 14. The process as claimed in claim 1,wherein the oxidizable substrate is selected from the group consistingof sodium thiocyanate (NaSCN), potassium thiocyanate (KSCN), sodiumbisulfite (NaHSO₃), sodium hydrosulfite (Na₂S₂O₄), sodium metabisulfite(Na₂S₂O₅), sodium nitrite (NaNO₂), potassium nitrite (KNO₂), sodiumhypochlorite (NaOCl) and potassium iodide (KI).
 15. The process asclaimed in claim 1, wherein the oxygen donor is hydrogen peroxide.
 16. Aprocess for enzymatically producing a treatment agent in a fluid statethat lacks an enzymatic catalyst, the treatment agent comprising atleast one oxygenated chemical species in a free state, the processcomprising: a) bringing into contact in an aqueous reaction bath amixture comprising: an enzymatic catalyst comprising a peroxidase,wherein the enzymatic catalyst is in a solid and divided phase, but in afree state, in the aqueous reaction bath; a flocculating agent selectedfrom the group consisting of polysaccharides, anionicheteropolysaccharides and polyacrylamines, and/or a coagulating agentselected from the group consisting of salts of aluminum and salts ofiron; an oxygen donor; and a substrate; b) generating an oxygenatedchemical species in the free state by allowing the enzymatic catalyst tocatalyze oxidation of the substrate by the oxygen donor; and c)separating the aqueous reaction bath into a first fraction enriched withthe enzymatic catalyst in the solid and divided phase, and a secondfraction that lacks the enzymatic catalyst and contains the treatmentagent, wherein the process is carried out in a reactor comprising fourcompartments, wherein two or three of said compartments are stirredcontinuously, the process comprising: i) receiving in a firstcompartment the enzymatic catalyst and the coagulating agent andoptionally a thickening agent, and stirring the first compartment at afirst speed; ii) in a second compartment, optionally receiving theflocculant and optionally receiving a pH-correcting agent, and stirringthe second compartment at a second speed, wherein the second speed isslower than the first speed; iii) receiving in a third compartment theoxidizable substrate and the oxygen donor, and stirring the thirdcompartment at a third speed, wherein the third speed is slower than thefirst speed; and iv) in a fourth compartment, separating the aqueousreaction bath into said first fraction enriched with the enzymaticcatalyst in solid and divided phase, and said second fraction thatcontains the treatment agent and is devoid of the enzymatic catalyst;wherein the aqueous bath is separated by a technique selected from thegroup consisting of decantation, flotation, centrifugation, filtrationand cyclonic separation.
 17. The process as claimed in claim 1, whereinthe second fraction is used to decontaminate the surfaces of alimentaryproducts.
 18. The process as claimed in claim 1, wherein the coagulatingagent is selected from the group consisting of aluminum sulfate,aluminum chloride, sodium aluminate, aluminum polyhydroxychloride,aluminum polyhydroxysulfate, aluminum polyhydroxychlorosulfate, basicaluminum polychlorosulfate, aluminum polyhydroxychlorosilicate, aluminumfluorosulfate, ferrous sulfate, ferric sulfate, ferric chloride andferric chlorosulfate.
 19. The process as claimed in claim 16, whereinthe coagulating agent is selected from the group consisting of aluminumsulfate, aluminum chloride, sodium aluminate, aluminumpolyhydroxychloride, aluminum polyhydroxysulfate, aluminumpolyhydroxychlorosulfate, basic aluminum polychlorosulfate, aluminumpolyhydroxychlorosilicate, aluminum fluorosulfate, ferrous sulfate,ferric sulfate, ferric chloride and ferric chlorosulfate.